Ig114 has two mutations in it. One is a conservative mutation, but the other is not. we are E/B transferring jtk2541 in pBca9523 into pMLL6-KA.
jtk2541 is {rbs.ffGFP}.
We will then combine jtk2541 in KA with ig114 in CK to produce ig114.jtk2541 in CA. This will enable us to easily check whether the AraC-Pbad promoter is still working and inducible.

Stage 1 parts grew up last night. Most things grew. Picked 4 colonies for most of the parts from stage 2 to miniprep tomorrow.
Yesterday we plated all the stage 1 parts and grew them up on plates. Today we picked 2 colonies for each to miniprep tomorrow.
Reassembled igem10_064 in KC and retransformed igem10_065 CK (Also picked colonies from an old plate).
Also retransformed Sbb18 in KA Lefty because miniprep is weak.

We labeled our transformation plate wrong so when we went to plate the cells many of them were plated on the wrong antibiotics. We still had colonies grow because they were co-transformed and had resistance to all antibiotics. This also explains why so many colonies grew when we tested for co-transformation. We have split our stage 1 parts into 4 groups:

1. Colonies grew on proper antibiotics and were not co-transformed. There are 12 of these. We mini-prepped these today and will do restriction mapping properly on Monday
2. Colonies that grew on wrong antibiotics need to be redone. There are 10 of these.We digested, ligated and transformed them today.
3. Parts that we thought we hadn't done (see yesterday's notes), but it turns out we did do them without realizing it since our plates were labeled wrong. Since we did them twice and they were all plated correctly the second time we will pick colonies, do Colony PCR, check for co-transformation and mini-prep these 9 on Monday.
4. Colonies that grew on the right antibiotics but were co-transformed. We will pick more colonies from the original plate to do ColPCR

We got cell growth for every part we plated (although in some parts there were very few colonies, about 3 or 4).
We made a Colony PCR Mastermix and tried to distribute it to two PCR plates with the robot but the robot kept giving us an error message (It said it couldn't pipette 19uL if there was 17uL in the tube but there was 2 ml in the tube and we wanted it to pipette 17uL). We gave up and I pipetted all 136 wells by hand.
Christoph and I picked colonies, added them to the PCR plate, innoculated them in a 96-well plate and left them in the incubator. We ran a ColPCR3K program on the both PCR plates.
Christoph noticed that we had based all of our csv robot files from the wrong list. We made all of the parts we needed but some of the parts had to be inserted into multiple vector backbones. We began making the 6 forgotten parts by hand. 3 of these parts need to be transformed into both Righties and Lefties so we're making 9 parts by hand.
We dabbed broth from our 96-well inoculated plate on both AKC plates and LB-only plates as negative and positive controls respectively for whether we picked any co-transformed colonies.
We need to run a huge gel when the PCR finishes at 4:40pm. Then we need to analyze the results. Christoph already named all the new composite parts and calculated their sizes.

To Do

We will mini-prep our good colonies tomorrow. Hopefully we'll have at least two good copies for each part so that we can continue with Plates A and B for stage 2.
We can start planning for Stage 2 assembly when we have free time. If we do it beforehand hopefully we'll catch any mistakes before running the robot
We should make a list of all the information Clothos gives us after it solves for the assembly tree. We've had to spent a good chunk of time writing excel formulas and making spreadsheets just so we know exactly how to assemble everything

While plating Stage1 2ab assembly set A we may have reversed the order of Col 1 B (30 ul).
The cells from plate A3-D3 may be in wells B4-B1 instead of B1-B4 on the 2x12 plated for Col 1A from the reaction plate.
Also note rows A and B were switched on the plate for Col 5A as noted on the plate. A is 30ul and B is 60ul instead of the other way around.

Redoing Stage1B and doing Stage1A 2ab assembly today.
Check autoclave at 4:30.
Decided to redo all of B along with A. We can do them together on the robot by doubling everything but changing the source plate
Made dilution plate A, and added 60uL of dilution to dilution plate B. 7 with 11 still looked different
Distributed digestion cocktail to both dilution plates Note: Robot begins pipetting air if it uses the same tip for two long. We stopped the program halfway through to change its tip Distributed Righty and Lefty parts to reaction plates. Made sure there were duplicate columns for parts being transformed twice Checked that at this point all wells had the same volume Put plates in thermocycler to incubate and heat kill Distributed antibiotics to 10 plates. All of LB agar was contaminated so will pour that during the rescue step added ligation mixture to each well and let incubate at room temperature.

Robot 2AB assembly Stage 1

Yesterday we did stage 1 of our 2AB assembly only on plate B. Today we will repeat the process on plate A without making all of the mistakes that we did yesterday.

June 15:
Autoclaved our strips for plating. Learned how to work the autoclave machine

Made a new plate layout for the reaction plate. Wells are organized by what strain the part needs to be transformed into to
MISTAKE: Did not listen to Tim and organized wells by column instead of rows. Rows are better because the plating strips have 2 rows and 12 columns
MISTAKE: Did not read procedure all the way through in detail and assumed that we could use a well for two transformations. Our dilution and reaction plate needs to have duplicates of the parts that go into both Righty and Lefty strains. We have to add the duplicates in either the dilution or reaction plate
Made the dilution plate B (30ul of DNA into each well + 30ul of NEB2 stock diluted by a factor of 5). Dilution plate had the same layout as methylated stock plate so we did it by hand. Next time, we should consider adding duplicates of the parts being transformed twice.
NOTE: 7 with 11 was frozen in our methylated stocks plate. This is the part that was mini-prepped separately the day before. All other wells weren't frozen.

Had the robot make reaction plate (distribute digestion mastermix to all wells, distribute all Lefty parts, mix, distribute all righty parts, mix)
While the reaction plate incubated in the black thermocycler (which also did heat kill of the digestion enzymes) we distributed the antibiotics to the 24-well strips. We made antibiotics (dilute stock by 10, add 1ul of dye). This part went fine except we should have labeled the plates before we put them on the robot. Added LB agar to the plates and let them dry next to the flame.
MISTAKE: forgot to make second plate for parts being transformed twice. Column 5 in reaction plate needed to go to two different plates. We made the extra plate by hand

Had robot add the ligation cocktail to the wells and let it incubate at room temperature. We began thawing our bacteria.
Did transformation of bacteria. We had enough volume to seed at least 60uL of bacteria per column.
MISTAKE: Did not realize that the protocol was for seeding 10uL of bacteria. We wanted to seed about 150uL. We should not have done the centrifugation and aspiration of the supernatant step.
MISTAKE: ejecting tips from multi-channel before releasing cells on plate. We lost 60uL of our cells from column 7.
MISTAKE: We split the volume in column 5 into column 9 (because column 5 had to be transformed into both righty and lefty strains). Thus we also did not have enough volume for those parts.
MISTAKE: not taking a short break after lunch to re-energize and prevent making silly mistakes

Reorganize destination plate based on L and R not on antibiotics. CHECK

AUTOCLAVE 24 well Strips! CHECK

Digesting (1hr) Check

Dilute NEB2 and DNA.

Make plates (2hr) Check

Cherry pick antibiotics Check

Heat kill (20 mins)Check

Ligation (30 mins)Check

Transform + Rescuing (1hr) Check

Plate (1hr)Check

Forgot to make two copies of the parts that are both lefty and righty. So, we just used half of ligation product of each.
Here is the layout of the transformation plate:
Column 5 is Lefty and was transformed with only half the ligation product.
Column 9 is righty and was also transformed with half the ligation product.

Next step is to transform each part into a Righty or Lefty strain.
Manually transformed parts from dilution plate into Righty and Lefty strains. Incubated at 5:10. Take out at 5:50.
Transformation plate has same layout as dilution plate below. Here is layout again:

Did a test run with the robot using our dilution csv file. Checked cloumn 4 it worked well.
Miniprepped pMLL5+Bca1152 and sent in for sequencing as iGem021.
Using robot to dilute 4ul of each vector+part stage 0 assembly into 76ul of water. See this csv file: Media:Stage0PartsDilution.csv

Source plate layout:

'

1

2

3

4

5

6

7

8

9

10

11

12

A

6+2

6+11

6+22

7+9

7+24

5+23

8+17

4+10

9+3

9+20

7+24

B

6+2

6+11

6+22

7+10

5+3

5+23

8+17

4+10

9+3

9+20

9+11

C

6+4

6+12

6+23

7+11

5+3

8+1

8+18

4+16

9+8

9+23

9+23

D

6+4

6+12

6+23

7+13

5+8

8+1

8+18

4+16

9+8

9+23

pMLL5+Bca1091 (L)

E

6+5

6+13

6+24

7+14

5+8

8+4

8+21

4+17

9+11

pMLL4+Bjh1882 (L)

F

6+5

6+13

6+24

7+19

5+15

8+4

8+21

4+17

9+11

pMLL6+Bjh2245

G

6+7

6+19

7+2

7+23

5+20

8+6

4+9

4+18

9+13

6 +24

6+12

H

6+7

6+19

7+5

7+24

5+20

8+6

4+9

4+18

9+13

7 + 23

5+25 (D)

Destination plate layout:

Columns 1-6 need to be transformed into Righty strains. Columns 7-12 need to be transformed into Lefty strains.

Currently in DH10B. Need to Eco/Bam transfer it into pMLL5-CK.
Put E/B digest of Bca1152 in DH10B into thermocycler at 12:10pm. Take out at 1:10pm and do small fragment zymo cleanup.
Set up ligation of Bca1152 and pMLL5 at 2:15. Transform at 2:45.
Heat-shock transformed Bca1152+pMLL5 into Jtk049. Plate at 4:15pm.
Plated Bca1152+pMLL5 into Jtk049. Growing up over night.
Sent in iGEM020 for sequencing(6 with 12C from working box to double check).